G-protein coupled receptors (GPCRs) are important targets for drug discovery since they are involved in a wide range of cellular signalling pathways and are implicated in many pathological conditions, such as cardiovascular and mental disorders, cancer and AIDS. Agonist binding to GPCRs facilitates guanine nucleotide exchange in G-proteins by catalysing the release of GDP from the α-subunit of the heterotrimeric GTP binding proteins (G-proteins) and allowing the binding of GTP which activates the G-proteins (Milligan, G., Trends in Pharm. Sci., (2003), 24, No 2, 87-90).
There is a continuing desire within the pharmaceutical industry to exploit GPCRs and orphan GPCRs as drug targets. Many methods have been used to measure GPCR activity and in vitro assays form an important part of high throughput screening strategies in the search for new GPCR-active ligands. Recently, there has been a desire to move towards sensitive, non-radioactive, and in particular homogeneous screening assays for GPCRs. A robust signal, in particular an optical signal that can be easily measured on a spectrophotometer, is an advantage. Thus, fluorescently-labelled non-hydrolysable GTP analogues have been developed for screening assays for GPCR-active ligands. U.S. Pat. No. 6,323,186 (Klaubert et al) relates to dipyrromethene-boron difluoride-substituted nucleotides in which the dipyrromethene-boron difluoride label is attached to a phosphate group of the nucleotide. McEwen et al (Analytical Biochemistry, (2001), 291, 109-117) synthesised three BODIPY GTPγS analogues (FL, 515, and TR), BODIPY FL GppNHp and BODIPY FL GTP compounds to study guanine nucleotide binding. There were reported variances in both fluorescence output as well as binding affinities compared to reference compounds, GTPγS and GppNHp upon binding to Gαo, with some analogues showing significant fluorescence increase upon binding. It was also shown that binding affinities also varied depending on the Gα subtype under study. In a separate study, Gille et al (Naunyn-Schmeideberg's Arch. Pharmacol., (2003), 368, 210-215), showed that BODIPY-FL-guanosine 5′-[γ-thio]triphosphate (B-GTPγS) and BODIPY-FL-guanosine 5′-[β,γ-imido]triphosphate (B-GppNHp) induced fluorescence changes upon binding to purified Gs/Gi-proteins and were suggested as probes for monitoring receptor-mediated G-protein activation. However, in the expression systems employed, these analogues were found to bind to receptor-Gαs/Gαi fusion proteins with 1,100-5,600-fold and 17-55-fold lower affinity than GTPγS and GppNHp, respectively. Their conclusion was that the steric bulk of the BODIPY group strongly reduces the affinity of GTPγS/GppNHp analogues for G-proteins.
An homogeneous GTP binding assay for G-protein coupled receptors based on time resolved FRET has previously been described (Frang, H., et al GTP binding assay for GPCRs based on TR-FRET, Poster PO 8123, Ninth Annual Society for Biomolecular Screening, Portland, Oreg., 21-25 September 2003). In this assay a biotinylated BioKey® peptide is employed that recognizes only the GTP bound form of the Gα subunit. The biotinylated peptide enables binding of streptavidin-europium in close proximity to an acceptor-labelled GTP, which is also bound to the Gα subunit. FRET occurs as a result of interaction between the streptavidin-europium (donor) and the fluorescently labelled GTP analogue (Alexa647-GTP).
A patent application entitled “Method for Measuring Binding of a Test Compound to a G-Protein Coupled Receptor” (Amersham Biosciences UK Limited) and filed on Sep. 30, 2004 as GB 0421693.3, now published as international application WO2006/035208, discloses a method for measuring test compound binding to a G-protein coupled receptor by means of fluorescence resonance energy transfer (FRET). FRET is a distance-related process in which the electronic excited states of two dye molecules interact without emission of a photon. See, Forster, T., “Intermolecular Energy Transfer and Fluorescence”, Ann. Physik., Vol. 2, p. 55, (1948). One result of this interaction is that excitation of a donor molecule enhances the fluorescence emission of an acceptor molecule and the fluorescence quantum yield of the donor is correspondingly diminished. By “donor”, it is meant that the dye moiety is capable of absorbing energy from light and emits light at wavelength frequencies which are at least partly within the absorption spectrum of the acceptor. By “acceptor”, it is meant that the dye moiety is capable of absorbing energy at a wavelength emitted by a donor dye moiety. For FRET to occur, suitably, the donor and acceptor dye molecules must be in close proximity (typically between 10-100 Å), since energy transfer efficiency decreases inversely as the 6th power of the distance (r) between the donor and acceptor molecules.
While a variety of fluorescent dye-nucleotide conjugates are available, the selection of a particular fluorescent label for use in a protein binding assay can be problematic, since the electronic and spatial requirements of the binding site of the protein of interest are difficult to predict a priori. There is therefore, still a requirement for new fluorescent GTP analogues that may be used for quantitating G-proteins and for studying the kinetics of agonist induced guanine nucleotide exchange in in vitro assays and in cellular systems.